Detection of intracellular RNA or DNA triggers unique sensors to activate the antiviral response: viral RNA activates the RIG-I-like receptor (RLR)-MAVS pathway, and foreign DNA activates the cGAS-STING pathway. These two antiviral pathways activate the type I interferon (IFN) response, which is essential for host defense against virus infection. However, chronic, dysregulated production of type I IFNs underlies a number of human autoimmune diseases, including systemic lupus erythematosus (SLE) and Aicardi-Goutieres Syndrome (AGS). Study of AGS in particular has revealed how cells carefully control endogenous nucleic acid metabolism to prevent aberrant activation of either the RLR-MAVS pathway or the cGAS-STING pathway. Whether additional, uncharacterized pathways exist that are also important for preventing inappropriate type I IFN production remains unknown. We have found that disruption of any of the components of the SUMOylation pathway results in a dramatic, spontaneous activation of the IFN response. We have discovered that SUMO-2 and SUMO-3, but not SUMO-1, are redundant for this IFN regulation, revealing a previously unknown and specific function of SUMO-2/3. Remarkably, this IFN response is independent of all known nucleic acid sensing pathways, revealing a novel mechanism that does not fit into the current paradigm of IFN response regulation. SUMO proteins are conjugated to target proteins in a manner that is analogous to ubiquitination, but our understanding of the functional outcomes of protein SUMOylation lags behind that of the ubiquitination system. In this grant, we will identify novel SUMO-2/3-modified proteins, test their role in regulation of the IFN response, and define the chromatin landscape of SUMO-2/3 binding. Together, our studies will provide the first detailed map of SUMO-2/3 regulation of the IFN response, with implications for human diseases caused by dysregulated IFN production.